1. Field of the Invention
This invention relates to methods and kits for performing specific binding assays. More particularly, the invention relates to a specific binding assay in which a luminescent substance, preferably the photoprotein, aequorin, is employed as a label. The luminescent specific binding assay of this invention is useful for determining the presence of various analytes in whole blood.
2. Description of the Background Art
Specific binding assay methods are used for quantitative and qualitative determinations of a wide variety of substances, generally referred to herein as "analytes." These substances may be large complex molecules, such as proteins, viruses, viral antigens, bacterial cells, cell surface receptors, enzymes, hormones, polysaccharides, glycoproteins, lipoproteins and the like, or small haptenic molecules, such as peptides, certain hormones, therapeutic drugs, drugs of abuse and the like.
These assays take advantage of specific binding reactions that occur between biological molecules. The specific binding reaction most commonly used is that which occurs between an antibody and an antigen. In that case, the specific binding assay is referred to as an immunoassay. Reactions between other binding pairs may also be employed. For example, interactions between enzymes and substrates, between hormones and receptors, and between complementary strands of nucleic acids, have been used for this purpose. Other binding reactions, such as those between avidin and biotin and between immunoglobulins and immunoglobulin binding proteins (e.g., Protein A and Protein G) have also been used advantageously in specific binding assays.
Specific binding assays may be configured in a variety of formats. For example, such assays may be competitive or sandwich assays. They may be homogeneous or heterogenous, and they may be sequential or simultaneous. In most specific binding assays, at least one of the members of the binding pair that participate in the specific binding reaction is labeled. The label provides a means for detecting and quantifying the reaction product. Radioimmunoassays employ a binding pair member (e.g., an antigen) that contains a radioactive isotope. In enzyme immunoassays, one of the binding pair members is labeled with an enzyme. The enzyme may, in turn, react with a substrate that produces a detectable signal, such as a color change.
Luminescent specific binding assays utilize any of a variety of chemiluminescent and bioluminescent labels. One such assay utilizes a photoprotein, known as aequorin, as the label. Aequorin is a high-affinity calcium ion-binding protein responsible for the bioluminescence of the jellyfish, Aequorea Victoria. Native aequorin is a photoprotein consisting of a single polypeptide chain of MW 21,000 daltons, containing one mole each of tightly bound coelenterate luciferin and oxygen. This complex is stable in the absence of calcium ions, and light emission is initiated upon the binding of three moles of calcium ions per mole of aequorin. In the presence of calcium ions, aequorin catalyzes the oxidation of luciferin to oxyluciferin with a concomitant flash of blue light (.lambda..sub.max =469 nm) which persists for approximately ten seconds. See, Stults, N. L. et al., Biochemistry 31, 1433-42 (1992) and references cited therein.
Aequorin can be isolated from Aequorea tissue. In addition, it can be produced by recombinant DNA techniques. See Cormier, M. J., U.S. Pat. No. 5,162,227 and Zenno. S. et al., U.S. Pat. No. 5,288,623. Modified forms of apoaequorin having enhanced bioluminescence properties have also been produced by recombinant DNA procedures. See Prasher, D., U.S. Pat. No. 5,360,728. As used herein, the term "aequorin" includes the native and recombinant forms of the photoprotein, as well as its modified forms as described in the aforementioned Prasher patent.
Other photoproteins are known that can be used as labels in specific binding assays. Such photoproteins include obeln, mnemiopsin, berovin, pholasin, luciferases and photoproteins isolated from Pelagia, Cypridina and ostracods.
Most specific binding assays are designed for analyzing biological fluids, such as blood or urine, for analytes of biological significance. When the goal of a specific binding assay is to determine the presence of a substance in whole blood, typically the sample must be pretreated to remove cellular components and hemoglobin, which can interfere with the assay. Specific binding assays are usually performed on the serum component of whole blood. Alternatively, whole blood assays have been designed to provide for a filtration or absorption step preceding the specific binding assay to minimize interference from cellular components and hemoglobin. See A Chen, F. M., U.S. Pat. No. 5,096,809. Herein, the terms "blood" and "whole blood" are used interchangeably and are defined to mean a homogeneous liquid that circulates through the body's cardiovascular channels that has not been treated to achieve any separation of its components, these components essentially comprising corpuscles, plasma, serum, and fibrin.
Often, it would be highly desirable to perform a specific binding assay directly on whole blood. Lower sample volumes are needed for assays performed directly on whole blood, because sample losses resulting from pretreatment steps are avoided. Moreover, the time for an assay to be conducted can be minimized if pretreatment steps are eliminated. In emergency room or operating room environments, particularly involving pediatric patients, these factors can be of critical importance.